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Observing the Microbial Cell
MULTIPLE CHOICE
1. Who compared ―small animals‖ from his teeth before and after drinking coffee?
a. Hooke
b. Fleming
c. Gram
d. Jenner
e. van Leeuwenhoek
ANS: E DIF: Easy REF: Introduction OBJ: Factual
TOP: Introduction
2. One __________ is one-thousandth of a millimeter.
a. micrometer
b. nanometer
c. meter
d. centi
e. kilo
ANS: A DIF: Medium REF: 2.1 OBJ: Factual
TOP: I.A
3. In humans, resolution is achieved by focusing an image on a retina, packed with light-absorbing:
a. rods
b. cones
c. nerves
d. photoreceptor cells
e. lenses
ANS: D DIF: Medium REF: 2.1 OBJ: Factual
TOP: I.A
4. Having fewer photoreceptors per surface area means higher:
a. resolution
b. magnification
c. refraction
d. reflection
e. wavelength
ANS: A DIF: Difficult REF: 2.1 OBJ: Applied
TOP: I.A
5. Resolution is the smallest distance by which two objects can be __________ and still be __________.
a. magnified; seen
b. separated; distinguished
c. magnified; separated
d. distinguished; separated
e. magnified; distinguished
ANS: B DIF: Medium REF: 2.1 OBJ: Applied
TOP: I.A.i
Observing the Microbial Cell | 281
6. A rod-shaped microbe is referred to as a:
a. bacillus
b. coccus
c. vibrio
d. strepto
e. spirochete
ANS: A DIF: Easy REF: 2.1 OBJ: Factual
TOP: I.C.ii.a
7. All electromagnetic radiation travels through a vacuum at what speed?
a. 3 × 108 mm/sec
b. 3 × 108
cm/sec
c. 3 × 108 m/sec
d. 3 × 108
ft/sec
e. 3 × 108 mph
ANS: C DIF: Difficult REF: 2.2 OBJ: Factual
TOP: II.A
8. If an object and its surroundings absorb or reflect radiation equally then the object will be:
a. undetectable
b. reflected
c. refracted
d. radiated
e. fluoresced
ANS: A DIF: Medium REF: 2.2 OBJ: Applied
TOP: II.A.ii.a
9. Which is the most important property that enables a lens to magnify an image?
a. absorption
b. fluorescence
c. reflection
d. refraction
e. scattering
ANS: D DIF: Medium REF: 2.2 OBJ: Applied
TOP: II.C
10. What is the key property that enables a lens to magnify an image?
a. reflection
b. resolution
c. frequency
d. refraction
e. wavelength
ANS: D DIF: Medium REF: 2.2 OBJ: Applied
TOP: II.C
282 | Chapter 2
11. Magnification without resolution is known as __________ magnification.
a. complete
b. zero
c. maximum
d. total
e. empty
ANS: E DIF: Easy REF: 2.2 OBJ: Factual
TOP: II.D
12. When two waves are out of phase by __________ wavelength, they produce destructive interference,
canceling each other’s amplitude and resulting in contrast in the image.
a. one-tenth
b. one-eighth
c. one-quarter
d. one-half
e. one
ANS: D DIF: Difficult REF: 2.2 OBJ: Factual
TOP: II.D
13. An image is magnified when light passes through a refractive material shaped so as to __________ its
rays.
a. absorb
b. block
c. concentrate
d. condense
e. spread
ANS: E DIF: Medium REF: 2.2 OBJ: Applied
TOP: II.D
14. Increasing the refractive index of the medium between the object and the objective lens increases:
a. refraction
b. reflection
c. magnification
d. resolution
e. wavelength
ANS: D DIF: Difficult REF: 2.2 OBJ: Applied
TOP: II.D
15. With the 100× lens, the refractive index of light passing through the specimen is maintained by
insertion of __________, with a refractive index comparable to that of glass.
a. immersion oil
b. water
c. air
d. bacteria
e. stain
ANS: A DIF: Easy REF: 2.3 OBJ: Factual
TOP: III.A
Observing the Microbial Cell | 283
16. As lens strength increases, the light cone __________ and the lens must be __________ the object.
a. narrows; nearer to
b. narrows; farther from
c. widens; nearer to
d. widens; farther from
e. widens; touch
ANS: C DIF: Medium REF: 2.3 OBJ: Factual
TOP: III.A
17. A/An __________ acts to vary the diameter of the light column in a light microscope.
a. condenser
b. objective
c. ocular
d. diaphragm
e. lens
ANS: D DIF: Easy REF: 2.3 OBJ: Factual
TOP: III.B.i
18. The total __________ of the microscope is obtained by multiplying the magnification of the ocular
lens by that of the objective lens.
a. resolution
b. magnification
c. refraction
d. reflection
e. wavelength
ANS: B DIF: Easy REF: 2.3 OBJ: Applied
TOP: III.B.i
19. Higher-power lenses require more light and thus an open:
a. ocular
b. lens
c. objective
d. condenser
e. diaphragm
ANS: E DIF: Easy REF: 2.3 OBJ: Applied
TOP: III.B.i
20. If you are using a microscope with a 10× ocular lens and a 100× objective, what is the total
magnification?
a. 10-fold
b. 100-fold
c. 110-fold
d. 1,000-fold
e. This is not enough information.
ANS: D DIF: Medium REF: 2.3 OBJ: Applied
TOP: III.B.i
284 | Chapter 2
21. Which is the counterstain in the Gram stain procedure?
a. crystal violet
b. methylene blue
c. malachite green
d. safranin
e. Gram’s iodine
ANS: D DIF: Easy REF: 2.3 OBJ: Factual
TOP: III.E.ii.a
22. Which of the following staining processes requires crystal violet?
a. acid-fast stain
b. antibody stain
c. negative stain
d. Gram stain
e. spore stain
ANS: D DIF: Easy REF: 2.3 OBJ: Factual
TOP: III.E.ii.a
23. Gram’s iodine is the __________ in the Gram staining procedure.
a. primary stain
b. counterstain
c. decolorizer
d. negative stain
e. mordant
ANS: E DIF: Easy REF: 2.3 OBJ: Factual
TOP: III.E.ii.a
24. Eukaryotes stain:
a. Gram-neutral
b. Gram-positive
c. Gram-negative
d. blue
e. no color
ANS: C DIF: Medium REF: 2.3 OBJ: Factual
TOP: III.E.ii.a
25. Which of the following is a true statement about Gram staining?
a. The Gram stain differentiates between the three domains.
b. In a Gram-negative cell, the crystal violet–iodide complex is retained by multiple layers of
peptidoglycan.
c. The outer membrane is disrupted by the decolorizer.
d. Human cells appear Gram-positive.
e. The mordant is used so that Gram-negative cells may be observed.
ANS: C DIF: Medium REF: 2.3 OBJ: Applied
TOP: III.E.ii.a
Observing the Microbial Cell | 285
26. The __________ in the Gram stain process binds to the crystal violet, generating a complex that is
held more tightly within the cell.
a. mordant
b. safranin
c. alcohol
d. bacteria
e. slide
ANS: A DIF: Medium REF: 2.3 OBJ: Applied
TOP: III.E.ii.a
27. Which of the following is a negative stain?
a. acid-fast stain
b. endospore stain
c. antibody stain
d. simple stain
e. capsule stain
ANS: E DIF: Easy REF: 2.3 OBJ: Factual
TOP: III.E.ii.d
28. X-ray diffraction and phase contrast microscopy both involve:
a. wave interference
b. observation of living specimens
c. differential stains
d. simple stains
e. shadowing.
ANS: A DIF: Difficult REF: 2.4 OBJ: Applied
TOP: IV.A | IV.B
29. Observations of bacterial flagella during motility are best suited to:
a. bright-field microscopy
b. dark-field microscopy
c. SEM
d. TEM
e. NMR
ANS: B DIF: Easy REF: 2.4 OBJ: Applied
TOP: IV.A.ii
30. A useful application of dark-field optics is the study of bacterial:
a. motility
b. surfaces
c. interiors
d. shape
e. structure
ANS: A DIF: Medium REF: 2.4 OBJ: Applied
TOP: IV.A.ii
286 | Chapter 2
31. In which type of microscopy do dust particles interfere the most?
a. bright-field microscopy
b. dark-field microscopy
c. phase-contrast microscopy
d. interference microscopy
e. fluorescence microscopy
ANS: B DIF: Easy REF: 2.4 OBJ: Conceptual
TOP: IV.A.iii
32. Which form of microscopy is based on the difference in refractive index between cell components and
the surrounding medium?
a. bright-field
b. dark-field
c. phase contrast
d. confocal
e. fluorescence
ANS: C DIF: Medium REF: 2.4 OBJ: Applied
TOP: IV.B
33. The digitally combined images of cryo-EM can achieve resolution comparable to that of:
a. SEM
b. TEM
c. interference microscopy
d. X-ray crystallography
e. dark-field microscopy
ANS: D DIF: Difficult REF: 2.4 OBJ: Conceptual
TOP: IV.D.i
34. When light is absorbed by an object and emitted at a longer wavelength, it is referred to as:
a. fluorescence
b. magnification
c. reflection
d. refraction
e. radiation
ANS: A DIF: Easy REF: 2.5 OBJ: Applied
TOP: V.A
35. Fluorescence requires excitation and emission at different:
a. refractive indexes
b. wavelengths
c. contrasts
d. refractions
e. densities
ANS: B DIF: Easy REF: 2.5 OBJ: Applied
TOP: V.A
Observing the Microbial Cell | 287
36. In fluorescence microscopy, incident light is absorbed by the specimen and reemitted at a __________
energy, resulting in a __________.
a. lower; longer wavelength
b. lower; shorter wavelength
c. higher; longer wavelength
d. higher; shorter wavelength
e. higher; higher contrast
ANS: A DIF: Difficult REF: 2.5 OBJ: Applied
TOP: V.A
37. The aromatic groups of the fluorophore DAPI associate exclusively with the:
a. cell wall
b. base pairs of DNA
c. flagella
d. cell membrane
e. pili
ANS: B DIF: Medium REF: 2.5 OBJ: Factual
TOP: V.B
38. The fluorophore acridine orange specifically binds:
a. cytoplasm
b. cell wall
c. protein
d. RNA
e. DNA
ANS: E DIF: Easy REF: 2.5 OBJ: Factual
TOP: V.B.i
39. The use of antibodies linked to fluorophores is known as:
a. fluorescence
b. immunofluorescence
c. X-ray diffraction
d. atomic force microscopy
e. cryo-EM
ANS: B DIF: Easy REF: 2.5 OBJ: Factual
TOP: V.B.ii
40. Which form of microscopy is used with DNA microarrays to observe differences in gene expression?
a. light microscopy
b. atomic force microscopy
c. SEM
d. TEM
e. confocal fluorescence microscopy
ANS: E DIF: Difficult REF: 2.5 OBJ: Applied
TOP: V.C
288 | Chapter 2
41. Which of the following is true of transmission electron microscopy but NOT scanning electron
microscopy?
a. The specimen is usually fixed and embedded.
b. The embedded specimen is cut into thin sections with a microtome.
c. The specimen is stained with heavy metal.
d. The specimen is viewed as three-dimensional.
e. The requirement for a vacuum precludes the viewing of live organisms.
ANS: B DIF: Medium REF: 2.6 OBJ: Applied
TOP: VI.A | VI.B
42. Transmission electron microscopy commonly has a resolution of __________ times the highest
resolution possible for light microscopy.
a. ten
b. one hundred
c. one thousand
d. ten thousand
e. one million
ANS: C DIF: Difficult REF: 2.6 OBJ: Applied
TOP: VI.A
43. In which one of the following types of microscopy is the specimen shadowed with heavy metal?
a. atomic force microscopy
b. SEM
c. TEM
d. X-ray diffraction
e. dark-field
ANS: B DIF: Medium REF: 2.6 OBJ: Factual
TOP: VI.A.ii
44. The knife used to cut embedded specimens for observation by TEM is called a:
a. crystallographer
b. microtome
c. grid
d. polymer
e. scalpel
ANS: B DIF: Easy REF: 2.6 OBJ: Factual
TOP: VI.B.i
45. Fixatives and heavy atom stains used in EM can introduce __________ into an image.
a. colors
b. resolution
c. refraction
d. artifacts
e. fluorescence
ANS: D DIF: Easy REF: 2.6 OBJ: Factual
TOP: VI.C
Observing the Microbial Cell | 289
46. A microscopic structure that is interpreted incorrectly is a/an:
a. microtome
b. crystal
c. shadow
d. antibody
e. artifact
ANS: E DIF: Easy REF: 2.6 OBJ: Factual
TOP: VI.C.i
47. In __________, because the frozen sample remains hydrated, the biological molecules retain the same
conformation as in solution.
a. cryo-EM
b. TEM
c. SEM
d. fluorescence microscopy
e. laser confocal microscopy
ANS: A DIF: Easy REF: 2.6 OBJ: Applied
TOP: VI.D.i
48. Atomic force microscopy measures __________ between a probe and an object to map the threedimensional topography of a cell.
a. hydrogen bonds
b. covalent interactions
c. van der Waals forces
d. pH changes
e. magnetic interactions
ANS: C DIF: Easy REF: 2.6 OBJ: Factual
TOP: VI.D.ii
49. Which type of microscopy is particularly useful to study the surfaces of live bacteria?
a. atomic force
b. SEM
c. TEM
d. dark-field
e. bright-field
ANS: A DIF: Easy REF: 2.6 OBJ: Applied
TOP: VI.D.ii
50. Which technique uses interference patterns from crystallized macromolecules to determine structure at
atomic resolution?
a. SEM
b. TEM
c. cryo-EM
d. X-ray diffraction analysis
e. atomic force microscopy
ANS: D DIF: Medium REF: 2.7 OBJ: Factual
TOP: VII.A.i
290 | Chapter 2
SHORT ANSWER
1. What does the phrase ―eagle-eyed‖ mean? Describe why it is scientifically accurate.
ANS:
Eagle-eyed means sharp-sighted. Eagles’ eyes can resolve things eight times as small or eight times as
far away as humans’ eyes because eagles’ photoreceptors are much more closely packed.
DIF: Medium REF: 2.1 OBJ: Conceptual TOP: I.A
2. Why is it possible to detect microbes but NOT resolve them?
ANS:
Detection is the ability to observe the presence of an object, such as when we detect a group of
bacteria in a culture tube. Even though we can detect the group, we can’t resolve individual cells
without magnification.
DIF: Easy REF: 2.1 OBJ: Applied TOP: I.B.i
3. List and describe three common shapes of bacteria.
ANS:
Bacilli (singular, bacillus) are rod-shaped bacteria. Cocci (singular, coccus) are spherical-shaped
bacteria. Spirochetes are tightly coiled spirals or corkscrew-shaped bacteria.
DIF: Easy REF: 2.1 OBJ: Factual TOP: I.C.ii
4. Explain when you would use Bacillus versus bacillus.
ANS:
Bacillus refers to a genus of organisms. A genus name is always capitalized and italicized. The term
bacillus refers to any rod-shaped microbe, which means that not all bacilli belong to the genus
Bacillus.
DIF: Easy REF: 2.1 OBJ: Applied TOP: I.C.ii
5. Describe three conditions that are necessary for electromagnetic radiation to resolve and object.
ANS:
There must be contrast between the object and its surroundings. The wavelength of the radiation must
be equal to or smaller that the size of the object. The detector must have sufficient resolution for the
given wavelength.
DIF: Medium REF: 2.2 OBJ: Factual TOP: II.A.ii
Observing the Microbial Cell | 291
6. List and briefly describe four ways that light interacts with objects.
ANS:
1. Absorption. Light energy is absorbed by an object. 2. Reflection. Wave front bounces off of object
at angle equal to its incident angle. 3. Refraction. Bending of light when it enters a substance that
slows its speed. 4. Scattering wave front interacts with object of smaller dimension than the
wavelength.
DIF: Medium REF: 2.2 OBJ: Applied TOP: II.B.i
7. Compare and contrast the radiation source, the lenses, and the image-capturing device in light and
TEM.
ANS:
The radiation source for light microscopy is a light, whereas for EM it is an electron source or tungsten
filament. The lenses in the light microscope are glass, whereas magnets are used in EM. The lenses
have similar functions and are arranged in the same order in both types of microscopy. Light
microscopy uses a condenser lens, whereas the lens in EM is called the projection lens. The imagecapturing device for light is the human eye, or sometimes a camera. The image-capturing device for
EM is a fluorescent screen.
DIF: Difficult REF: 2.3 | 2.6 OBJ: Conceptual TOP: III.B | VI.A
8. Describe the lens system of a compound microscope including the location of each lens and the
purpose of each.
ANS:
The condenser lens is above the light source and functions to concentrate light rays up through the
specimen. The objective lenses are immediately above the specimen and the function is to magnify the
specimen. A typical light microscope has objective lenses that magnify 10×, 40×, and 100×. The
ocular lens is located in the eyepiece and also magnifies the specimen. A typical ocular lens magnifies
10×.
DIF: Easy REF: 2.3 OBJ: Applied TOP: III.B.i
9. Why are stains used in microscopy? Compare and contrast the stains used in light versus electron
microscopy.
ANS:
Stains are used to increase the contrast between an object and its surroundings, so as to make it visible.
The stains used in light microscopy usually are charged and interact with different cellular
components. Positively charged dyes bind to negatively charged cell surfaces. They also are colored,
so they impart color to a cell or its components. The stains used for EM are heavy metals or salts,
which increase the density of certain components, again increasing contrast. In EM, the image of the
microbe is always black and white.
DIF: Difficult REF: 2.3 | 2.6 OBJ: Conceptual TOP: III.D | VI.B
292 | Chapter 2
10. Briefly explain why or how fixatives and stains used in microscopy may introduce artifacts. How
might this be determined?
ANS:
A specimen may be fixed in many different ways depending on the type of microscopy, but two
examples are heat and chemical fixation. Heat can denature certain cellular components and chemicals
can also alter structural integrity. Most stains are charged and interact with various cellular
components. These interactions may also lead to alteration in structural characteristics. Sometimes it
can be determined if something is an artifact by comparing the results using different forms of
microscopy.
DIF: Difficult REF: 2.3 OBJ: Conceptual TOP: III.D
11. Compare and contrast simple and differential stains. Give examples of each.
ANS:
A simple stain is used to increase contrast in order to visualize a specimen. Methylene blue, for
example, will stain all cells equally. A differential stain allows one to distinguish between cell types or
characteristics. The Gram stain procedure is a differential stain. It can be used to distinguish between
Gram-positive and Gram-negative cells.
DIF: Medium REF: 2.3 OBJ: Applied TOP: III.E
12. Briefly state the steps in the Gram stain procedure. What would happen if you accidentally forgot to
use the counterstain?
ANS:
The Gram stain procedure involves four steps:
1. the primary stain—crystal violet
2. the mordant—Gram’s iodine
3. the decolorizer—ethanol
4. the counterstain—safranin
If you left out the counterstain step it would be difficult to detect any Gram-negative microbes that
were present in the specimen.
DIF: Medium REF: 2.3 OBJ: Conceptual TOP: III.E.ii.a
13. Why do some bacteria appear purple after being Gram stained and other appear red?
ANS:
Gram-negative cells have a few layers of peptidoglycan cell wall and an outer lipopolysaccharide
membrane. Gram-positive organisms have several layers of peptidoglycan and no outer membrane.
The multiple layers of peptidoglycan retain the crystal violet–iodine complex so appear purple. Gramnegative cells do not retain the crystal violet because there are few layers of peptidoglycan and the
outer membrane is disrupted by the decolorizer.
DIF: Medium REF: 2.3 OBJ: Conceptual TOP: III.E.ii.a
Observing the Microbial Cell | 293
14. Explain how the modified condenser in dark-field microscopy is used to make small microbes visible.
Give one advantage and one disadvantage of dark-field microscopy.
ANS:
The condenser contains an opaque disk held by three ―spider legs‖ across an open ring. No light
travels directly up through the specimen so the only light that reaches the eye is light that is scattered
by objects on the slide. This scattered light allows detection of objects that are too small to be resolved
by light rays. Advantages include the ability to detect live organisms without staining, detection of
very small organisms that can’t be seen with bright-field microscopy, and ability to visualize motility
of microbes. Disadvantages are that shapes of objects can’t be easily resolved and particulates may be
mistaken for organisms.
DIF: Medium REF: 2.4 OBJ: Applied TOP: IV.A
15. Explain phase-contrast microscopy and give one advantage and one disadvantage of phase-contrast
microscopy.
ANS:
Phase-contrast microscopy exploits differences in refractive index between cell components and
transforms them into differences in intensity of transmitted light. Advantages are that live cells can be
viewed and the organelles of eukaryotes are visible. A disadvantage is that it is less effective for
organisms whose cytoplasm as a low refractive index.
DIF: Medium REF: 2.4 OBJ: Applied TOP: IV.B
16. Define a fluorophore and give three examples of how they can be used to label cells.
ANS:
A fluorophore is a fluorescent molecule that can be used to stain a specimen for observation with a
fluorescence microscope. Some fluorophores, such as DAPI, have affinity for certain cell chemicals.
Antibodies can be labeled with fluorescent dyes and reacted with specific targets in
immunofluorescence. Short sequences of DNA attached to a fluorophore can be used to hybridize to
and label target DNA.
DIF: Difficult REF: 2.5 OBJ: Applied TOP: V.B
17. How does laser scanning confocal microscopy produce images?
ANS:
A laser beam is focused onto the specimen and scanned across it in two planes at right angles to each
other. The laser beam excites a fluorophore and both the excitation and emitted light are focused
together. This results in images with very high resolution.
DIF: Medium REF: 2.5 OBJ: Applied TOP: V.C
294 | Chapter 2
18. Give a few reasons why living organisms may NOT be observed by TEM or SEM.
ANS:
In TEM, the specimens are fixed and embedded into a polymer for sectioning. The specimen is then
stained with heavy metal to increase contrast. In SEM, the entire organism is shadowed with heavy
metal prior to observation. Most importantly, however, the entire optical column of the EM must be
maintained under vacuum, and a living specimen would be quickly destroyed by an electron beam.
DIF: Easy REF: 2.6 OBJ: Conceptual TOP: VI.A | VI.B
19. Why is it that a photographic image from an electron microscope is black and white?
ANS:
The original image is produced when the electrons bombard a fluorescent screen. The resultant image
is processed by a computer to appear as black and white with intensities in the entire range of grays in
between.
DIF: Difficult REF: 2.6 OBJ: Conceptual TOP: VI.A
20. Describe three methods of sample preparation for electron microscopy.
ANS:
1. Samples can be embedded in a polymer and cut into thin sections with a microtome, then coated
with a heavy metal. 2. Samples can be sprayed onto a copper grid then treated with a heavy metal. 3.
Samples may be flash frozen for cryo-EM.
DIF: Medium REF: 2.6 OBJ: Applied TOP: VI.B